Poor delivery of oxygen and nutrients to engrafted cells is a central challenge in engineered tissues for regenerative medicine, and improper microvascularization (too little or too much) underlies the etiology of a dizzying array of human diseases. To enhance knowledge on the mechanism of microvascular development in engineered tissues, technologies enabling control over the local 3D microenvironments of microvessels would enable direct testing of candidate hypotheses. Native microvessels exhibit structures such as basement membrane, open lumens, and expression of apical markers and proteoglycans, and form networks with spatial architectures that are well defined and intimately related to tissue function. Current approaches for mimicking microvessel microenvironments fall short of these goals, as microfabrication techniques based on coating open channels with endothelial cells exhibit low correspondence to the structures of native microvessels, and homogeneous seeding of endothelial cells inside a bulk 3D scaffold does not allow flexible network design. Hence, there currently remains a critical technological gap in addressing the central question: "How does the local 3D microenvironment affect the development of microvascular networks in engineered tissues?" The long-range goals of this grant are to gain knowledge of how blood vessels form, and improve the viability of engineered tissues through enhanced vascularization. The immediate objective of this grant is to study how the local microenvironment affects microvascular development in engineered tissues. Our approach leverages a set of microfabrication techniques developed in our laboratory, including a collagen-interfacing method for forming stable multiphase hydrogels. These methods aim to preserve native-like structure of microvessels and allow for control of spatial architecture of the microvascular network. After optimizing the collagen-interfacing method to work with both naturally derived and synthetic hydrogels, we will aim to test two hypotheses (in vitro and in vivo) about the role of local microenvironment in the development of microvessels. PUBLIC HEALTH RELEVANCE: The goal of this R01 grant is to study microvascularization with control over the local tissue environment. It will leverage a new set of 3D tissue microfabrication technologies that allow spatially controlled patterning of multiple phases of extracellular matrices.